Expanding control of the tumor cell cycle with a CDK2/4/6 inhibitor.

The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2- breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G1 kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.

Discovery of PF-06873600, a CDK2/4/6 Inhibitor for the Treatment of Cancer.

Control of the cell cycle through selective pharmacological inhibition of CDK4/6 has proven beneficial in the treatment of breast cancer. Extending this level of control to additional cell cycle CDK isoforms represents an opportunity to expand to additional tumor types and potentially provide benefits to patients that develop tumors resistant to selective CDK4/6 inhibitors. However, broad-spectrum CDK inhibitors have a long history of failure due to safety concerns. In this approach, we describe the use of structure-based drug design and Free-Wilson analysis to optimize a series of CDK2/4/6 inhibitors. Further, we detail the use of molecular dynamics simulations to provide insights into the basis for selectivity against CDK9. Based on overall potency, selectivity, and ADME profile, PF-06873600 (22) was identified as a candidate for the treatment of cancer and advanced to phase 1 clinical trials.

Spectrum and Degree of CDK Drug Interactions Predicts Clinical Performance.

Therapeutically targeting aberrant intracellular kinase signaling is attractive from a biological perspective but drug development is often hindered by toxicities and inadequate efficacy. Predicting drug behaviors using cellular and animal models is confounded by redundant kinase activities, a lack of unique substrates, and cell-specific signaling networks. Cyclin-dependent kinase (CDK) drugs exemplify this phenomenon because they are reported to target common processes yet have distinct clinical activities. Tumor cell studies of ATP-competitive CDK drugs (dinaciclib, AG-024322, abemaciclib, palbociclib, ribociclib) indicate similar pharmacology while analyses in untransformed cells illuminates significant differences. To resolve this apparent disconnect, drug behaviors are described at the molecular level. Nonkinase binding studies and kinome interaction analysis (recombinant and endogenous kinases) reveal that proteins outside of the CDK family appear to have little role in dinaciclib/palbociclib/ribociclib pharmacology, may contribute for abemaciclib, and confounds AG-024322 analysis. CDK2 and CDK6 cocrystal structures with the drugs identify the molecular interactions responsible for potency and kinase selectivity. Efficient drug binding to the unique hinge architecture of CDKs enables selectivity toward most of the human kinome. Selectivity between CDK family members is achieved through interactions with nonconserved elements of the ATP-binding pocket. Integrating clinical drug exposures into the analysis predicts that both palbociclib and ribociclib are CDK4/6 inhibitors, abemaciclib inhibits CDK4/6/9, and dinaciclib is a broad-spectrum CDK inhibitor (CDK2/3/4/6/9). Understanding the molecular components of potency and selectivity also facilitates rational design of future generations of kinase-directed drugs. Mol Cancer Ther; 15(10); 2273-81. ©2016 AACR.

Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance.

Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform.

Ack1: activation and regulation by allostery.

The non-receptor tyrosine kinase Ack1 belongs to a unique multi-domain protein kinase family, Ack. Ack is the only family of SH3 domain containing kinases to have an SH3 domain following the kinase domain; others have their SH3 domains preceding the kinase domain. Previous reports have suggested that Ack1 does not require phosphorylation for activation and the enzyme activity of the isolated kinase domain is low relative to other kinases. It has been shown to dimerize in the cellular environment, which augments its enzyme activity. The molecular mechanism of activation, however, remains unknown. Here we present structural and biochemical data on Ack1 kinase domain, and kinase domain+SH3 domain that suggest that Ack1 in its monomeric state is autoinhibited, like EGFR and CDK. The activation of the kinase domain may require N-lobe mediated symmetric dimerization, which may be facilitated by the N-terminal SAM domain. Results presented here show that SH3 domain, unlike in Src family tyrosine kinases, does not directly control the activation state of the enzyme. Instead we speculate that the SH3 domain may play a regulatory role by facilitating binding of the MIG6 homologous region to the kinase domain. We postulate that features of Ack1 activation and regulation parallel those of receptor tyrosine kinase EGFR with some interesting differences.

KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients.

Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors. Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance.

CYP3A4 is a vitamin D-24- and 25-hydroxylase: analysis of structure function by site-directed mutagenesis.

Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1 alpha-hydroxyvitamin D(2) (1 alpha OHD(2)) as substrate. The ratio of rates of 24-hydroxylation of 1 alpha-hydroxyvitamin D(3) (1 alpha OHD(3)), 1 alpha OHD(2), and vitamin D(2) by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D(2), 1,24(S)-dihydroxyvitamin D(2), and 1,24-dihydroxyvitamin D(3) were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1 alpha OHD(2) by CYP3A4 correlated significantly with 6 beta-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, alpha-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1 alpha OHD(2) and 1 alpha OHD(3) were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D(2), 1 alpha OHD(2), and 1 alpha OHD(3).

Activation of the anticancer prodrugs cyclophosphamide and ifosfamide: identification of cytochrome P450 2B enzymes and site-specific mutants with improved enzyme kinetics.

Cyclophosphamide (CPA) and ifosfamide (IFA) are oxazaphosphorine anticancer prodrugs metabolized by two alternative cytochrome P450 (P450) pathways, drug activation by 4-hydroxylation and drug inactivation by N-dechloroethylation, which generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde. CPA and IFA metabolism catalyzed by P450s 2B1, 2B4, 2B5, and seven site-specific 2B1 mutants was studied in a reconstituted Escherichia coli expression system to identify residues that contribute to the unique activities and substrate specificities of these enzymes. The catalytic efficiency of CPA 4-hydroxylation by rat P450 2B1 was 10- to 35-fold higher than that of rabbit P450 2B4 or 2B5. With IFA, approximately 50% of metabolism proceeded via N-dechloroethylation for 2B1 and 2B4, whereas CPA N-dechloroethylation corresponded to only approximately 3% of total metabolism (2B1) or was absent (2B4, 2B5). Improved catalytic efficiency of CPA and IFA 4-hydroxylation was obtained upon substitution of 2B1 Ile-114 by Val, and replacement of Val-363 by Leu or Ile selectively suppressed CPA N-dechloroethylation >or=90%. P450 2B1-V367A, containing the Ala replacement found in 2B5, exhibited only approximately 10% of wild-type 2B1 activity for both substrates. Canine P450 2B11, which has Val-114, Leu-363, and Val-367, was therefore predicted to be a regioselective CPA 4-hydroxylase with high catalytic efficiency. Indeed, P450 2B11 was 7- to 8-fold more active as a CPA and IFA 4-hydroxylase than 2B1, exhibited a highly desirable low K(m) (80-160 microM), and catalyzed no CPA N-dechloroethylation. These findings provide insight into the role of specific P450 2B residues in oxazaphosphorine metabolism and pave the way for gene therapeutic applications using P450 enzymes with improved catalytic activity toward these anticancer prodrug substrates.

Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of P450 conformations and the coordination of redox partner binding.

A 1.9-A molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B' and C, the N terminus of helix I, and the beta(4) region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F' and G' helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding.

Topological changes in the CYP3A4 active site probed with phenyldiazene: effect of interaction with NADPH-cytochrome P450 reductase and cytochrome b5 and of site-directed mutagenesis.

The active site topology of heterologously expressed CYP3A4 purified from an Escherichia coli expression system was examined using phenyldiazene. Incubation of CYP3A4 with phenyldiazene and subsequent oxidation yielded all four potential N-phenylprotoporphyrin IX regioisomers derived from attack on an available nitrogen atom in pyrrole rings B, A, C, or D (N(B):N(A):N(C):N(D) = 6:73:7: 13). Further study using 28 active site mutants showed that substitution of residues closer to the heme, Ala-305, Thr-309, or Ala-370, with a larger residue caused the most drastic changes in regioisomer formation, which reflected the location of each amino acid residue replaced in a CYP3A4 homology model. Previous studies have suggested a conformational change in CYP3A4 upon binding of NADPH-cytochrome P450 reductase (CPR) or cytochrome b(5) (b(5)). Therefore, regioisomer formation was also compared in the absence of redox partners and in the presence of CPR, b(5), or both. Formation of all four regioisomers in CYP3A4 wild type, particularly the minor ones, was reduced in the presence of b(5). CPR also greatly decreased the three minor isomers but increased the major isomer significantly. The presence of b(5) and CPR restored minor isomer formation and suppressed the enhancement of N(A) formation caused by CPR alone. Interestingly, the effects of the redox partners differed among representative active site mutants. In particular, the increase in N(C) upon substitution of Ala-370 with Phe was significantly reversed in the presence of redox partners, strongly suggesting that a conformational change occurs around pyrrole ring C due to protein-protein interactions between CYP3A4 and CPR or b(5).

An open conformation of mammalian cytochrome P450 2B4 at 1.6-A resolution.

The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-A crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the alpha-helical and beta-sheet domains without perturbing the overall P450 fold. This cleft is primarily formed by helices B' to C and F to G. The conformation of these regions is dramatically different from that of the other structurally defined mammalian P450, 2C5/3LVdH, in which the F to G and B' to C regions encapsulate one side of the active site to produce a closed form of the enzyme. The open conformation of 2B4 is trapped by reversible formation of a homodimer in which the residues between helices F and G of one molecule partially fill the open cleft of a symmetry-related molecule, and an intermolecular coordinate bond occurs between H226 and the heme iron. This dimer is observed both in solution and in the crystal. Differences between the structures of 2C5 and 2B4 suggest that defined regions of xenobiotic metabolizing P450s may adopt a substantial range of energetically accessible conformations without perturbing the overall fold. This conformational flexibility is likely to facilitate substrate access, metabolic versatility, and product egress.

Analysis of homotropic and heterotropic cooperativity of diazepam oxidation by CYP3A4 using site-directed mutagenesis and kinetic modeling.

The structural basis for the cooperativity of diazepam oxidation catalyzed by human cytochrome P450 3A4 (CYP3A4) and 40 mutants has been investigated. An ordered two-site model in which substrates bind first to a catalytic/effector site and then to the catalytic site was used to explain sigmoidal kinetics for temazepam formation but hyperbolic kinetics for nordiazepam formation. In this model diazepam binds to the enzyme-substrate complex with a greater affinity (K(S2)=140 microM) than to free enzyme (K(S1)=960 microM). Residues 107, 119, 211, 301, 304, 309, 369, 370, and 373 play an important role in determining regioselectivity of diazepam oxidation. Interestingly, S119F and A370F displayed sigmoidal kinetics for nordiazepam formation, whereas I301F exhibited hyperbolic kinetics for both products. In the presence of increasing concentrations of testosterone, K(S1) for diazepam decreased, whereas K(S2) increased. The data suggest that three sites exist within the active pocket.

Site-directed mutagenesis of cytochrome P450eryF: implications for substrate oxidation, cooperativity, and topology of the active site.

The role of five active-site residues (Phe-78, Gly-91, Ser-171, Ile-174, and Leu-175) has been investigated in P450eryF, the only bacterial P450 known to show cooperativity. The residues were selected based on two-ligand-bound P450eryF structures and previous mutagenesis studies of other cytochromes P450. To better understand the role of these residues in substrate catalysis and cooperativity, each mutant was generated in the wild-type and A245T background, a substitution that enables P450eryF to oxidize testosterone and 7-benzyloxyquinoline (7-BQ). Replacement of Phe-78 with tryptophan decreased cooperativity of 9-aminophenanthrene binding, with little effect on testosterone binding or oxidation. Interestingly, substitution of Gly-91 with alanine or phenylalanine abolished the type-I spectral change elicited by testosterone and significantly decreased testosterone hydroxylation. However, G91A/A245T showed a 4-fold higher k(cat) value with 7-BQ compared with A245T. Replacement of Ser-171 with alanine or phenylalanine did not alter cooperativity of testosterone binding but significantly decreased binding affinity and oxidation of testosterone and 7-BQ. The only mutant that exhibited an increased testosterone binding affinity and increased rates of testosterone and 7-BQ oxidation was I174F. Substitution of Ile-175 with phenylalanine decreased testosterone and 7-BQ oxidation. Reaction with phenyldiazene showed that P450eryF may be much more open above pyrrole ring B than other cytochromes P450 and indicated significant changes in active-site topology in some of the mutants. The study suggests a crucial role of residues Ser-171, Ile-174, and Leu-175, which are part of a distal ligand site, in addition to the proximal Gly-91 in determining the oxidative properties of P450eryF.